Human cytomegalovirus (HCMV) infection of primary cells results in massive alterations in normal cellular metabolism and regulation of cellular pathways that govern cell death, nucleic acid replication, cellular membrane homeostasis, cytoskeleton integrity, and intracellular trafficking of proteins. Numerous studies have documented the widespread dysregulation of cellular gene transcription following HCMV infection arguing that major changes in cellular phenotypes are controlled at a transcriptional level. However, crude regulation of essential and highly coordinated cellular functions such as protein transport and membrane homeostasis could result in a cellular environment that would also limit the production of infectious virus. Our recent studies have demonstrated that HCMV miRNAs regulate the secretory/endocytic pathway during infection by targeting genes encoding-key components of these pathways, thus providing another layer of HCMV regulation of cellular functions. Regulation of these pathways by HCMV miRNA results in inhibition of the release of pro-inflammatory cytokines and the reorganization of intracellular membranes to facilitate efficient production of infectious virus. In this project, the specific genes in the secretory/endocytic pathway targeted by HCMV miRNAs will be identified by a comprehensive approach combining PAR-CLIP methodology with informatics followed by rigorous validation. Initially, the role of validated miRNA targets in the regulation of trafficking in the endocytic an secretory pathways will be defined, followed by investigation of the role of these miRNA targets in cytokine trafficking and secretion, membrane reorganization, and infectious virus assembly. Validation of the role of these cellular targets of HCMV miRNAs in regulation of the secretory/endocytic pathways of the infected cells and their phenotypic effects on cytokine secretion and virus assembly will be accomplished by engineering recombinant HCMVs expressing specific combinations of shRNAs targeting genes within these pathways. It is anticipated that these studies will elucidate the role of these regulatory RNAs in the outcome of HCMV infection of the cell as well as defining a new and novel mode of regulation of essential cellular pathways leading to a cellular environment optimized for assembly of infectious virions.